Functioning nanomachines seen in real-time in living bacteria using single-molecule and super-resolution fluorescence imaging.
Int J Mol Sci 12 (2011) 2518-2542
Molecular machines are examples of "pre-established" nanotechnology, driving the basic biochemistry of living cells. They encompass an enormous range of function, including fuel generation for chemical processes, transport of molecular components within the cell, cellular mobility, signal transduction and the replication of the genetic code, amongst many others. Much of our understanding of such nanometer length scale machines has come from in vitro studies performed in isolated, artificial conditions. Researchers are now tackling the challenges of studying nanomachines in their native environments. In this review, we outline recent in vivo investigations on nanomachines in model bacterial systems using state-of-the-art genetics technology combined with cutting-edge single-molecule and super-resolution fluorescence microscopy. We conclude that single-molecule and super-resolution fluorescence imaging provide powerful tools for the biochemical, structural and functional characterization of biological nanomachines. The integrative spatial, temporal, and single-molecule data obtained simultaneously from fluorescence imaging open an avenue for systems-level single-molecule cellular biophysics and in vivo biochemistry.
Using bespoke fluorescence microscopy to study the soft condensed matter of living cells at the single molecule level
Journal of Physics: Conference Series 286 (2011)
Using bespoke fluorescence microscopy to study the soft condensed matter of living cells at the single molecule level
CONDENSED MATTER AND MATERIALS PHYSICS CONFERENCE (CMMP10) 286 (2011)
A general approach for segmenting elongated and stubby biological objects: Extending a chord length transform with the radon transform
2010 7th IEEE International Symposium on Biomedical Imaging: From Nano to Macro, ISBI 2010 - Proceedings (2010) 161-164
Automatic, high-throughput, quantification of the precise position and orientation of biological objects is essential for studying living, biomedically relevant processes from timelapse microscopy images. These measurements frequently include precise estimates for the center-of-mass as well as the location of the true object boundaries e.g. the membranes of cells. This paper describes a region-oriented segmentation approach applied to the detection of both insects at the mm length scale as well as bacteria at the μm length scale. Despite the differences in length scale, images of both objects have similar aspect ratio, and it is common to have overlapping objects in images of both. This thus presents a challenge for any segmentation algorithm. Our approach performs all orientation detection through a chord length transform, so the task of separating overlapping objects in a two-dimensional image is reformulated as a voxel-labeling problem within a three-dimensional volume. It then utilizes the directional information from the Radon transformed image. Experimental results in simulation show that our method is effective in separating clustered elongated but stubby objects with aspect ratios not far from 1. The applications in detecting insects and Escherichia coli bacteria demonstrate the value of our approach. ©2010 IEEE.
Proceedings of the National Academy of Sciences of the United States of America 107 (2010) 11347-11351
Shining the spotlight on functional molecular complexes: The new science of single-molecule cell biology.
Commun Integr Biol 3 (2010) 415-418
Single-molecule research is emerging as one of the fastest growing fields within the biosciences. Historically, most of the techniques employed have operated largely in the world of the test tube in which the components of the biological system under investigation have been extracted and purified from cells to reduce them to just the key ingredients under study, and this research has involved novel, pioneering methods of biophysics to obtain single-molecule measurements. What has emerged recently is the technical ability to now perform key single-molecule experiments whilst retaining the native biological context-namely to do single-molecule experiments on functional living cells. This presents essentially a new science of "single-molecule cell biology", which combines classical cell biology approaches with modern single-molecule biophysics. Here, key recent studies which have pushed back the boundaries of this field are discussed.
Science 328 (2010) 498-501
The multiprotein replisome complex that replicates DNA has been extensively characterized in vitro, but its composition and architecture in vivo is unknown. Using millisecond single-molecule fluorescence microscopy in living cells expressing fluorescent derivatives of replisome components, we have examined replisome stoichiometry and architecture. Active Escherichia coli replisomes contain three molecules of the replicative polymerase, rather than the historically accepted two. These are associated with three molecules of tau, a clamp loader component that trimerizes polymerase. Only two of the three sliding clamps are always associated with the core replisome. Single-strand binding protein has a broader spatial distribution than the core components, with 5 to 11 tetramers per replisome. This in vivo technique could provide single-molecule insight into other molecular machines.
Proc Natl Acad Sci U S A 107 (2010) 11347-11351
Most biological processes are performed by multiprotein complexes. Traditionally described as static entities, evidence is now emerging that their components can be highly dynamic, exchanging constantly with cellular pools. The bacterial flagellar motor contains approximately 13 different proteins and provides an ideal system to study functional molecular complexes. It is powered by transmembrane ion flux through a ring of stator complexes that push on a central rotor. The Escherichia coli motor switches direction stochastically in response to binding of the response regulator CheY to the rotor switch component FliM. Much is known of the static motor structure, but we are just beginning to understand the dynamics of its individual components. Here we measure the stoichiometry and turnover of FliM in functioning flagellar motors, by using high-resolution fluorescence microscopy of E. coli expressing genomically encoded YPet derivatives of FliM at physiological levels. We show that the approximately 30 FliM molecules per motor exist in two discrete populations, one tightly associated with the motor and the other undergoing stochastic turnover. This turnover of FliM molecules depends on the presence of active CheY, suggesting a potential role in the process of motor switching. In many ways the bacterial flagellar motor is as an archetype macromolecular assembly, and our results may have further implications for the functional relevance of protein turnover in other large molecular complexes.
Proc Natl Acad Sci U S A 106 (2009) 11582-11587
Many bacterial species swim by employing ion-driven molecular motors that power the rotation of helical filaments. Signals are transmitted to the motor from the external environment via the chemotaxis pathway. In bidirectional motors, the binding of phosphorylated CheY (CheY-P) to the motor is presumed to instigate conformational changes that result in a different rotor-stator interface, resulting in rotation in the alternative direction. Controlling when this switch occurs enables bacteria to accumulate in areas favorable for their survival. Unlike most species that swim with bidirectional motors, Rhodobacter sphaeroides employs a single stop-start flagellar motor. Here, we asked, how does the binding of CheY-P stop the motor in R. sphaeroides--using a clutch or a brake? By applying external force with viscous flow or optical tweezers, we show that the R. sphaeroides motor is stopped using a brake. The motor stops at 27-28 discrete angles, locked in place by a relatively high torque, approximately 2-3 times its stall torque.
A novel multiple particle tracking algorithm for noisy in vivo data by minimal path optimization within the spatio-temporal volume
Proceedings - 2009 IEEE International Symposium on Biomedical Imaging: From Nano to Macro, ISBI 2009 (2009) 1158-1161
Automated tracking of fluorescent particles in living cells is vital for subcellular stoichoimetry analysis [1, 2]. Here, a new automatic tracking algorithm is described to track multiple particles, based on minimal path optimization. After linking feature points frame-by-frame, spatio-temporal data from time-lapse microscopy are combined together to construct a transformed 3D volume. The trajectories are then generated from the minimal energy path as defined by the solution of the time-dependent partial differential equation using a gray weighted distance transform dynamic programming method. Results from simulated and experimental data demonstrate that our novel automatic method gives sub-pixel accuracy even for very noisy images. © 2009 IEEE.
J Vis Exp (2009) 1508-
Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular level. To date the shear complexity of biological systems has caused precise single-molecule experimentation to be far too demanding, instead focusing on studies of single systems using relatively crude bulk ensemble-average measurements. However, many important processes occur in the living cell at the level of just one or a few molecules; ensemble measurements generally mask the stochastic and heterogeneous nature of these events. Here, using advanced optical microscopy and analytical image analysis tools we demonstrate how to monitor proteins within a single living bacterial cell to a precision of single molecules and how we can observe dynamics within molecular complexes in functioning biological machines. The techniques are directly relevant physiologically. They are minimally-perturbative and non-invasive to the biological sample under study and are fully attuned for investigations in living material, features not readily available to other single-molecule approaches of biophysics. In addition, the biological specimens studied all produce fluorescently-tagged protein at levels which are almost identical to the unmodified cell strains ("genomic encoding"), as opposed to the more common but less ideal approach for generating significantly more protein than would occur naturally ('plasmid expression'). Thus, the actual biological samples which will be investigated are significantly closer to the natural organisms, and therefore the observations more relevant to real physiological processes.
Millisecond timescale slimfield imaging and automated quantification of single fluorescent protein molecules for use in probing complex biological processes.
Integr Biol (Camb) 1 (2009) 602-612
Fluorescence microscopy offers a minimally perturbative approach to probe biology in vivo. However, available techniques are limited both in sensitivity and temporal resolution for commonly used fluorescent proteins. Here we present a new imaging system with a diagnostic toolkit that caters for the detection and quantification of fluorescent proteins for use in fast functional imaging at the single-molecule level. It utilizes customized microscopy with a mode of illumination we call "slimfield" suitable for rapid (approximately millisecond) temporal resolution on a range of common fluorescent proteins. Slimfield is cheap and simple, allowing excitation intensities approximately 100 times greater than those of widefield imaging, permitting single-molecule detection at high speed. We demonstrate its application on several purified fluorescent proteins in standard use as genetically-encoded reporter molecules. Controlled in vitro experiments indicate single protein molecules over a field of view of approximately 30 microm(2) area, large enough to encapsulate complete prokaryotic and small eukaryotic cells. Using a novel diagnostic toolkit we demonstrate automated detection and quantification of single molecules with maximum imaging rates for a 128 x 128 pixel array of approximately 500 frames per second with a localization precision for these photophysically poor fluorophores to within 50 nm. We report for the first time the imaging of the dim enhanced cyan fluorescent protein (ECFP) and CyPet at the single-molecule level. Applying modifications, we performed simultaneous dual-colour slimfield imaging for use in co-localization and FRET. We present preliminary in vivo imaging on bacterial cells and demonstrate approximately millisecond timescale functional imaging at the single-molecule level with negligible photodamage.
Proceedings of the National Academy of Sciences of the United States of America 106 (2009) 11582-11587
Journal of Visualized Experiments (2009)
Biochemical Society Transactions 36 (2008) 1032-1036
Clustering and dynamics of cytochrome bd-I complexes in the Escherichia coli plasma membrane in vivo
Molecular Microbiology 70 (2008) 1397-1407
Variable stoichiometry of the TatA component of the twin-arginine protein transport system observed by in vivo single-molecule imaging.
Proc Natl Acad Sci U S A 105 (2008) 15376-15381
The twin-arginine translocation (Tat) system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. The essential components of the Tat pathway are the membrane proteins TatA, TatB, and TatC. TatA is thought to form the protein translocating element of the Tat system. Current models for Tat transport make predictions about the oligomeric state of TatA and whether, and how, this state changes during the transport cycle. We determined the oligomeric state of TatA directly at native levels of expression in living cells by photophysical analysis of individual yellow fluorescent protein-labeled TatA complexes. TatA forms complexes exhibiting a broad range of stoichiometries with an average of approximately 25 TatA subunits per complex. Fourier analysis of the stoichiometry distribution suggests the complexes are assembled from tetramer units. Modeling the diffusion behavior of the complexes suggests that TatA protomers associate as a ring and not a bundle. Each cell contains approximately 15 mobile TatA complexes and a pool of approximately 100 TatA molecules in a more disperse state in the membrane. Dissipation of the protonmotive force that drives Tat transport has no affect on TatA complex stoichiometry. TatA complexes do not form in cells lacking TatBC, suggesting that TatBC controls the oligomeric state of TatA. Our data support the TatA polymerization model for the mechanism of Tat transport.
Biologist 55 (2008) 33-39
Proteins, so small that one billion would fit on a full stop, carry out most of the vital activities in living cells; they drive chemical reactions, transport cargoes, communicate with the outside world and even segregate chromosomes. A novel approach now allows us to monitor single proteins in complicated molecular machines, and it seems that biological components wear out and get replaced just as they do in man-made machines.
Potential for disulphide-bridge formation and phosphorylation-dependent modification of mechanical properties in the N2B-Domain of human cardiac titin
BIOPHYSICAL JOURNAL (2007) 522A-523A
BIOPHYS J (2007) 527A-527A